Throughout this specification, the term “particle” refers to the constituents of liquid sample aliquots that may be molecules of varying types and sizes, nanoparticles, virus like particles, liposomes, emulsions, bacteria, colloids, etc. Their size range may lie between 1 nm and several thousand micrometers.
Solutions containing solutes such as molecules, viruses, nanoparticles, liposomes, etc. are often analyzed after their constituent fractions are separated by liquid chromatography technique such as size exclusion chromatography (SEC), which is also referred to as high performance liquid chromatography (HPLC) or another separation technique such as field flow fractionation (FFF), hydrophobic interaction chromatography (HIC), or ion exchange chromatography (IEX). Such measurements may include determination of solute concentration, solution viscosity, and light scattering properties. The latter measurement used in combination with a corresponding concentration determination may be used to derive the size, molar mass, aggregation, and associations of the solutions constituent elements. To improve these measurements the light scattering detection is frequently performed by measuring the light scattered by the separated sample at a plurality of angles with respect to the direction illuminating light beam. This technique of measuring the intensity of the light scattered by a liquid sample as a function of angle is referred to as multiangle light scattering (MALS).
MALS measurements may also be performed in a “batch mode” wherein an unfractionated, prepared liquid sample contained within a scintillation vial or cuvette is placed into the path of the illuminating beam. An alternative to the traditional batch measurement wherein the sample is injected, unfractionated into a flow cell is generally referred to as “stop-flow”or “microbatch” mode. In microbatch mode, after a measurement is made, the sample is removed from the flow cell by an injection of another sample or solvent through the flow cell inlet. The present invention is equally relevant to both microbatch and the standard flow-through measurements discussed above.
While flow through MALS cells have taken many forms through the years, the ease and reliability of MALS measurements took a dramatic step forward with the introduction of an axial flow cell described by Phillips, et. al. in U.S. Pat. No. 4,616,927 (issued Oct. 14, 1986). The basic structure of the axial cell assembly as described by Phillips is shown in FIG. 1. A right circular glass cylinder 101 contains a small polished bore 102 drilled through a diameter about midway between the cylinder's base and top. Flow through fixtures 103 and 108 contain a channel 104 through which a liquid sample may pass. These fixtures also house optical windows 105 which are held into the fixtures by retaining elements 106. A seal is maintained between the window 105 and the channel 104 by a gasket or o-ring. Fluid passes from the inlet fixture 103 through a connection tube 107 that directs the sample to flow through the cylindrical flow cell 101 and then through the exit fixture 108. From there the sample may flow to waste or another detector or a sample reclamation system. A light beam 109, generally from a laser source, is directed to pass through the optical windows, 105 along the same path as the liquid sample. This entire assembly 100 was then placed into a read head with spaces milled therein rigidly hold the elements in place as well as possible. In general, a plurality of photodetectors (not shown) are also rigidly held within the read head positioned circumferentially about the center of the flow cell. These photodetectors gather light scattered from the light beam by the sample as it passes through the bore 102. Once the flow cell assembly was fitted into the read head, the laser was aligned such that the beam 109 passes through the center of the bore 102 without grazing its walls.
A problem associated with all flow through optical cells, and in particular those which measure static light scattering, is the inevitable presence of contaminants accumulating within the cell itself. These contaminants can be introduced from various sources, including detritus shed upstream detectors or preparative systems, such as chromatography columns, or they can be accidentally introduced by direct injection in mircobatch measurements. Even the samples themselves may contribute to dirtying the cell by forming aggregates with a strong affinity for the internal optical systems. Once a cell is contaminated, it must be cleaned, either in situ by flushing or more aggressive means such as sonication, as described by Trainoff in U.S. Pat. No. 6,452,672 B1 (Issued Sep. 17, 2002), or by removing the cell glass itself and performing a manual cleaning. While in situ cleaning techniques can be effective in the short term, most MALS cells must be removed and cleaned manually on a somewhat regular basis. Flow cell cleaning usually requires some expertise and extreme caution to be certain none of the inner or outer surfaces are soiled by contaminants such as fingerprints, residues and particulates. Further, damage to the cell can occur both during manual cleaning, and albeit less frequently, during normal instrument operation. It is therefore inevitable that the flow cell will need to be removed from the assembly from time to time.
One limitation of the Phillips cell is the difficulty associated with realignment of its optical elements after disassembly for cleaning or other maintenance. In the Phillips system, the optical axis is defined according to the position of at least three elements, the inlet and outlet fixtures 103 and 108, each of which house windows through which the beam will pass, and the bore 102 of the flow cell 101 itself. If any of these items are in misalignment, the optical system may fail. What is more, the position of the connection elements 107 at least partly define the height of the bore relative to the windows, so any wear or issues associated with orientation of the cylindrical connection elements may cause the entire bore to be canted. For these reasons every time the cell is removed from the instrument, every optical element in the chain must be realigned to ensure the beam is reliably able to pass through both windows and the bore of the flow cell without grazing any interfaces. Further, the lack of any orienting or registration elements on the glass cell itself contributed to the possibility of misalignment due to placing the cell in 180° from its originally aligned position, as well the possibility of play in the connection elements allowing the bore to be positioned non-parallel to the optical axis defined by the position of the windows. These limitations made realignment of the Phillips cell cumbersome every time the cell was removed from the system.
These problems relating to effectively reproducing the alignment conditions of the cell relative to the beam when the cell was removed were addressed by Janik, et. al, in U.S. Pat. No. 5,404,217 (Issued Apr. 4, 1995), hereinafter referred to as the '217 patent. Janik describes a rigidly connected flow cell manifold housing all elements that may then be removed as a single unit from the optical bench of the MALS instrument. As shown in FIG. 2, the flow cell 201 not contains two flattened surfaces 202 at the inlet and outlet of the bore. These surfaces serve to register the cell and prevent any rotation thereof relative to the inlet and outlet manifold halves 203 and 204. One embodiment of the Janik invention includes a step 205 milled into the glass cell 201 itself. This step is pressed up against either pins or a seat present in the manifold halves, further ensuring consistent directional alignment of the bore with the manifold halves. Each manifold half in turn contains an inlet or outlet 206 milled into the top surface. Fluiding bearing tubing is connected to these ports by means of an appropriate fitting. One of these ports 206 directs the sample via a fluid channel to the bore 207, parallel thereto, of the flow cell 201. The flow cell is sandwiched between the manifold halves and sealed thereto by gaskets or o-rings. Fluid leaving the other end of the bore is directed to the outlet port 206 and exists the instrument to proceed to waste, another analysis instrument or a sample recover system. Each manifold half also contains an optical window that allows a laser beam to pass along the fluid path in the manifold halves and through the flow cell bore. As in the Phillips invention, Janik directs a laser beam through the window within the inlet manifold half 203, and emerging from the window in the outlet manifold half 204 after passing through the bore of the flow cell without grazing any surfaces contained therein. The manifold halves are held together by bolts, and are rigidly connected to a base plate 208. Once fully assembled, the flow cell manifold system is placed within the read head of a MALS instrument to which the assembly had previously been aligned. This method facilitates the reassembly of the instrument after flow cell cleaning and provides reliable reproducibility of alignment for a particular manifold assembly with a particular flow cell and its associated MALS instrument. Thus Janik enabled a user to disassemble and reassemble the flow cell system without the need to realign the laser after each cleaning.
In all cases discussed thus far, it should be noted that each element of the optical system, in particular the flow cell itself, was mated with the other elements for the life of the alignment. Thus, in all cases, before the present invention it was not possible to replace a flow cell with a different cell without the need to realign the system. Of course the realignment of an optical system adds further complexity to the entire process and is generally performed only by well trained personnel, generally requiring the shipment of the entire system back to the manufacturer. It is an objective of the present invention to enable the replacement of one optical flow cell with another within the same MALS instrument without the need to optically realign the system. It is a further objective of the invention to enable the end user to reliably replace the flow cell without specialized equipment or knowhow. It is another objective of the invention to facilitate the use of distinct flow cells with specific properties, such as refractive index differences or varying bore widths within a MALS instrument. Another objective of the invention is to enable Process Analytic Technology (PAT) to monitor reactions by utilizing bore widths compatible with this technology and permitting the replacement of flow cells online without the need to halt the reaction system.